Molecularly engineered cells created to study the impact of specific genes on cell physiology are fundamental tools for biomedical discovery. A major focus of contemporary cancer research is the study of the molecular mechanisms underlying cell survival and replication by creation of cell lines that express tumor suppressor genes or have oncogene knockouts. While critical for the study of the uncontrolled replication that characterizes cancer cells, these cell lines are extremely difficult to generate and maintain due to their growth disadvantage. New technologies based on microfabricated cell arrays have the potential to provide an enabling platform for routine creation and maintenance of these cellular tools. In the current grant application, an interdisciplinary team will develop arrays of releasable cell rafts to overcome the technical challenges in isolation and maintenance of clonal colonies with a growth disadvantage. Easily implemented and inexpensive fabrication techniques will be used to manufacture the arrays. Array design and materials will be optimized for transfection and expansion of cells on the array. Critical biological controls will be performed to confirm cell health, viability, and clonality. Methods for array analysis and selection of clonal colonies will be developed based on standard microscopic imaging. A simple mechanical system will be optimized in order to release individual rafts on which target cells reside. This work will include the design and testing of hardware and software to enable semi-automated analysis and cell isolation. Cell lines needed for studies of tumor suppressor biology will be created and maintained, and the technology will be validated by comparison with conventional approaches. The fundamental innovations developed in this research will decrease the costs of array manufacture and will produce a flexible, reliable and simple to use technology. The capabilities engendered by this new method for cell cloning will lead to new insights into the control of cancer cell growth and survival.